Reactive oxygen species‐responsive dual‐targeted nanosystem promoted immunogenic cell death against breast cancer

Abstract The development of an optimal treatment modality to improve the therapeutic outcome of breast cancer patients is still difficult. Poor antigen presentation to T cells is a major challenge in cancer immunotherapy. In this study, a synergistic immunotherapy strategy for breast cancer incorporating immune cell infiltration, immunogenic cell death (ICD), and dendritic cell (DC) maturation through a reactive oxygen species (ROS)‐responsive dual‐targeted smart nanosystem (anti‐PD‐L1‐TKNP) for the simultaneous release of DOX, R848, and MIP‐3α in the tumor microenvironment is reported. Following local injection, anti‐PD‐L1‐DOX‐R848‐MIP‐3α/thioketal nanoparticle (TKNP) converts tumor cells to a vaccine owing to the combinatorial effect of DOX‐induced ICD, R848‐mediated immunostimulatory properties, and MIP‐3α‐induced immune cell recruitment in the tumor microenvironment. Intratumoral injection of anti‐PD‐L1‐DOX‐R848‐MIP‐3α/TKNP caused significant regression of breast cancer. Mechanistic studies reveal that anti‐PD‐L1‐DOX‐R848‐MIP‐3α/TKNP specifically targets tumor tissue, resulting in maximum exposure of calreticulin (CRT) and HMGB1 in tumors, and significantly enhances intratumoral infiltration of CD4+ and CD8+ T cells in tumors. Therefore, a combined strategy using dual‐targeted ROS‐responsive TKNP highlights the significant application of nanoparticles in modulating the tumor microenvironment and could be a clinical treatment strategy for effective breast cancer management.


| INTRODUCTION
Dendritic cells (DCs), as professional antigen-presenting cells (APCs), are crucial in cancer immunotherapy by presenting antigens to cytotoxic T cells. 1 Although DCs are effective APCs, insufficient Toll-like signaling leads to poor antigen presentation to T cells.Immunoadjuvants such as Toll-like receptor (TLR) agonists induce Toll-like signaling and are vital in DC maturation. 2 Resiquimod (R848), a TLR7/8 agonist which causes DC maturation, has a potent antitumor effect in skin cancer and advanced leukemia patient. 3merous clinical trials have investigated the immunogenic cell death (ICD) phenomenon exerted by chemotherapeutic agents. 4cent studies have demonstrated that anticancer agents such as oxaliplatin and doxorubicin (DOX) alter the tumor microenvironment and cause ICD of tumor cells. 5ICD is characterized by calreticulin (CRT) exposure on the cell surface, high-mobility group box 1 (HMGB1) release from the cell nucleus, and adenosine triphosphate (ATP) secretion, which provides an "eat me" signal for cytotoxic T-cell activation. 6spite its significant therapeutic benefit, ICD has failed to produce a robust anticancer effect because of the nonspecificity or low-dose distribution in the tumor tissue encountered with the free chemotherapeutic drugs. 7spite the large application of immunoadjuvant and chemotherapeutic agents, cancer immunotherapy still faces challenges because of the insufficient infiltration of immune cells in the tumor microenvironment. 8Chemokine therapy can address this issue as it enhances immune cell recruitment in the tumor microenvironment. 9Chemokines such as macrophage inflammatory protein-3 alpha (MIP-3α) bind with the CCR6 receptor expressed on DCs and cause DC recruitment at the inflammation site. 10mbination therapy with chemotherapeutic agents, immunoadjuvants, and chemokines has a robust antitumor effect in cancer immunotherapy. 11However, combination therapy has a poor pharmacokinetic profile, nonspecificity, and the activation of pro-inflammatory cascade leading to serious side effects. 12 address this issue, tumor microenvironment-responsive nanoparticles were rationally designed for combination chemoimmunotherapy. 13,14Benefiting from the high difference in reactive oxygen species (ROS) level between normal and tumor cells (100-fold), we constructed ROS-responsive thioketal nanoparticles (TKNPs) for better tumor specificity. 15Various ROS-responsive moieties such as thioketals, peroxalates, and selenium-containing linkages are being used for constructing ROS-responsive nanoparticles. 16However, thioketal linkage was employed in our study for constructing ROS-responsive nanoparticles because of its better stability to enzymatic degradation. 17Furthermore, we sought to develop a dual-targeted nanosystem by conjugating anti-PD-L1 antibodies on the ROS-responsive nanoparticle surface to enhance cellular internalization.Our engineered nanosystem was further co-encapsulated with DOX, R848, and MIP-3α to elicit tumor-specific immune response.Our approach of co-encapsulation preserves the immunological activities of chemotherapeutic agents, immunoadjuvants, and chemokines, thereby providing maximal therapeutic outcomes. 18In the high ROS condition of the tumor microenvironment, TKNPs convert into hydrophilic sulfoxides and sulfones, enabling the release of therapeutic cargos in the tumor microenvironment. 19We believe that our optimized nanosystem will prime CD8 + T cells and demonstrate a strong antitumor effect against local and metastatic tumors, thereby having a great impact on the development of personalized cancer immunotherapy (Figure 1).
Dynamic light scattering (DLS; Brookhaven Instruments Corporation, Holtsville, NY) suggested that the TKNP hydrodynamic diameter was 94.5 ± 1.4 nm, while the polydispersity indices (PDI) and average zeta potential were 0.28 and 36.9 ± 1.9 mV, respectively (Figure 2e).The detailed result of optimization of hydrodynamic diameter, loading capacity (LC), and encapsulation efficiency (EE) with different mass ratios for the synthesis of DOX, MIP-3α, and R848-loaded TKNP is described in Supporting Information.The optimized thioketal polymer, DOX, R848, and MIP-3α in the mass ratio of 10:1:0.5:0.04,respectively, was selected which showed a hydrodynamic diameter of 101.0 ± 1.5 nm, PDI of 0.26, and zeta potential of À33.3 ± 1.2 mV as measured using DLS (Figure 2e,f).

| Tailoring of anti-PD-L1 antibody on TKNP using DPEMA
DPEMA provides an active site for the adhesion of anti-PD-L1 antibody to the TKNP surface via Schiff's base substitution or Michael addition reaction.The particle size of the optimized anti-PD-L1 antibody anchored on TKNP was 103.3 ± 3.0 nm, while the PDI was 0.28, and the zeta potential was À10.5 ± 0.3 mV (Figure S3).
The amount of anti-PD-L1 antibody adhered on the surface of 1 mg TKNP was 111.6 ± 8.5 μg (Figure 2h), and the conjugation efficiency (CE) was 93.0 ± 7.1% (Figure 2i).Stability results also suggested that anti-PD-L1-DOX-R848-MIP-3α/TKNP was highly stable in PBS for 7 days with no change in the particle size and the particle size was similar (104.1 ± 3.4 nm) to PDI-0.29 (Figure S4).The Supporting Information describes detailed results regarding optimization of the anti-PD-L1 antibody amount conjugated on TKNP (Figure S5).

| Oxidative stress-mediated release of DOX and R848
DOX and R848 are released from the TKNP surface owing to the oxidative stress-mediated degradation of TKNP (Figure 2j,k).Following incubation for 120 h at an acidic pH, the release of DOX and R848 was approximately 41% and 35%, respectively.Incubation at neutral pH for 120 h caused an approximately 25% and 24% release of DOX and R848, respectively.However, following incubation with 0.1 mM H 2 O 2 for up to 120 h, the release of DOX and R848 was approximately 82% and 70%, respectively.Cumulative release of DOX and R848 in response to oxidative stress was attributed to nanoparticle degradation.

| Cellular internalization study of nanoparticles and DOX in 2D and 3D models
When compared to TKNP alone, anti-PD-L1-TKNP treatment resulted in increased internalization of nanoparticles (2.7-fold) and DOX (2.6-fold) in the MDA-MB-231 cell line, as determined using fluorescence-activating cell sorting (FACS) analysis.However, there was no significant difference in the cellular internalization of nanoparticles and DOX in nontargeted and anti-PD-L1-targeted TKNP in the BT-20 cell line (Figure 3a).S6).Thus, this study also further highlights the PD-L1 receptor-mediated cellular internalization of anti-PD-L1-DOX-TKNP.

| Tumor retention assay
To evaluate tumor retention and targeting ability of TKNP and anti-PD-L1-TKNP, In Vivo Imaging System (IVIS) was used, and tumor retention of the nanoparticles was evaluated at 0, 0.5, 6, 12, and 24 h.Subsequently, we sacrificed the mice at 24 h to perform ex vivo imaging of the vital organs.Superior retention in the tumor zone was obtained following anti-PD-L1-TKNP treatment (43.7 ± 7.8%) compared to TKNP (29.8 ± 2.8%) alone (Figure 5c,d).There was no accumulation in vital organs such as the heart and lungs, suggesting nontoxicity of the nanoparticles.
There was a significant reduction in body weight in the free DOX and DOX-R848 MIP-3α-treated groups compared to the control.However, body weight was similar to the control in the DOX-R848-MIP-3α/TKNP and anti-PD-L1-DOX-R848-MIP-3α/ TKNP-treated groups.Furthermore, the tumor weight was significantly reduced (Figure 5i) in mice treated with anti-PD-L1-DOX-R848-MIP-3α/TKNP which was further confirmed using the morphological tumor images (Figure 5j).Furthermore, ICD provides an "eat me signal" which eventually leads to DC maturation, thereby causing antigen processing and presentation to T cells. 20DCs has a pivotal role in the activation of cellular immunity.However, cellular immunity is impaired by the poor antigen presentation by DCs. 21DCs follows TLR signaling pathway and assist in the antigen presentation to T cells. 22Immunoadjuvants such as R848 play a significant role in DC maturation.Thus, we further examined the expression of surface molecules such as CD86 + on CD11c + MHCII + DCs derived from lymph nodes to assess DC maturation using a flow cytometer (Figure 5m).In the DOX-R848-MIP-3α/ TKNP-treated mice, the CD86 + ratio on CD11c + MHCII + DCs was significantly increased to 49.7 ± 5.5%, which was 1.8-fold (p < 0.01) higher than of DOX-R848-MIP-3α (27.6 ± 5.3%) cocktail.Additionally, maximum increment in the CD86 + ratio (74.3 ± 9.6%) on CD11c +- MHCII + DCs was noticed in anti-PD-L1-DOX-R848-MIP-3α/TKNPtreated mice, which was 2.7-fold (p < 0.001) and 1.5-fold (p < 0.01) higher than that of DOX-R848-MIP-3α and DOX-R848-MIP-3α/ TKNP, respectively.Increased DC maturation is accredited to the tumor microenvironment-specific release of R848 offered by our nanosystem.

| Infiltration of CD4 + and CD8 + T cells in the tumor microenvironment
It is well reported that increased ICD phenomenon and DC maturation lead to increased intratumoral infiltration of CD4 + and CD8 + T cells. 23us, we evaluated CD4 + and CD8 + T cells expression in the tumor tissue following immunofluorescence staining.CLSM was used to determine the intratumoral infiltration of CD4 + and CD8 + T cells (Figure 6a).Maximum expression of CD4 + and CD8 + (green and red fluorescence, respectively) in the tumor section of anti-PD-L1-DOX-R848-MIP-3α/TKNP-treated mice compared to that in other groups suggested maximum intratumoral infiltration of immune cell.
Similarly, we also evaluated the quantitative expression of intratumoral infiltration of CD4 + and CD8 + T cells after treatment with different formulations using flow cytometry (Figure 6b).The gating strategy is further provided in Supporting Information (Figure S11).
Secreted cytokines play a crucial role in boosting cellular immunity.Therefore, we measured cytokine levels such as IL-6 and TNF-α in mice sera at days 15 and 20 using Elisa assay (Figure 6d).Compared to the PBS group, increased serum IL-6 and TNF-α levels were observed on days 15 (60.9 ± 22.3 pg/ml and 56.7 ± 12.6 pg/ml) and 20 (60.7 ± 23.3 pg/ml and 54.8 ± 13.8 pg/ml), respectively, in the DOX-treated group.Compared to the DOX-R848-MIP-3α cocktail, DOX-R848-MIP-3α/TKNP increased the IL-6 and TNF-α levels at days 15 (135.7 ± 16.9 pg/ml; 1.6-fold and 142.5 ± 34.7 pg/ml; 1.7-fold) and 20 (131.2 ± 14.3 pg/ml; 1.5-fold and 131.8 ± 8.4 pg/ml; 1.6-fold), respectively.The incorporation of immunoadjuvant, chemokines, and cytotoxic agents in nanoparticles provides a better anticancer effect, causing an increase in cytokine levels.A more significant increment in the serum IL-6 and TNF-α levels were observed in anti-PD-L1-DOX-R848-MIP-3α/TKNP at days 15 (198.7 ± 28.1 pg/ml and 207.8 ± 25.9 pg/ml) and 20 (196.8 ± 22.9 pg/ml and 208.9 ± 25.9 pg/ml), respectively, which is accredited to the PD-L1-mediated tumor specificity and ROS-responsive release of therapeutic cargos.Clinical data on breast cancer have reflected durable therapeutic response following activation of patient's immune system. 24FDA approved atezolizumab for PD-L1-positive advanced breast cancer. 25wever, the proportion of breast cancer patients responding to phase Ib clinical trial of atezolizumab was only 12%. 26Major challenges in the immunotherapeutic approach are restricted to an inappropriate dose of cytotoxic drugs in the tumor microenvironment, poor antigen presentation, and poor infiltration of cytotoxic T lymphocytes in the tumor microenvironment. 27,28To combat the tumor heterogeneity with activated immunity, and strengthen the efficacy, complex formulations with several agents target different limitations of the therapeutic strategies is necessary. 29As a solution to the inappropriate dose of cytotoxic drugs, we used PD-L1-targeted and ROSresponsive nanoparticles enabling the administration of a concentrated dose of chemotherapeutic agents in the tumor site.The poor antigenic presentation was overcome by the nano-enabled release of R848 in the tumor microenvironment.To address poor infiltration of cytotoxic T lymphocytes, we also introduced chemokine therapy by encapsulating MIP-3α in our nanoparticles which accelerates immune cell migration in the tumor microenvironment.We have used MDA-MB-231 30,31 and 4T1 31 cell line for our study as they have been reported for high PD-L1 expression.However, BT-20 cell which expresses low PD-L1 32 expressions than MDA-MB-231 cell line was used for the evaluation of the targeting effect of anti-PD-L1-DOX-R848-MIP-3α/TKNP.Furthermore, we have also mentioned the rationale behind the usage of these cell lines in the manuscript as well.
Despite immunological silence of DOX, several clinical and preclinical evidence has suggested the emergence of DOX-induced ICD phenomenon. 33In a clinical setting, low dose of DOX has been reported to induce ICD.However, off-target effect and poor selectivity limit the clinical application of DOX-induced ICD. 6 In this study, we demonstrate the use of ROS-responsive and PD-L1-targeted nano-enabled chemotherapy for robust ICD induction in a xenograft 4T1 breast cancer model, an outcome that was not well exerted by free DOX.
Despite immune cell activation via ICD induction, cancer cells still evade their destruction by suppressing immune cells. 34To address this issue, we focused on the maturation of antigen-presenting cells such as DCs via pharmacological activation of TLR using TLR agonist (R848), which further instructs cytotoxic T cells to eradicate tumor cells.R848, which shares a similar structure with the FDA-approved TLR7/8 agonist imiquimod, is reported to be 100 times more potent. 2stemic administration of R848 exerts antitumor immune responses but a high dose is required for optimal therapeutic effect. 35Local injection of R848 combined with chemotherapeutic agents has shown complete tumor regression with long-term therapeutic effects.Yin et al. 36 reported that a 3 mg/kg dose of R848 was administered intraperitoneally nine times in 4T1 tumor-bearing mice model; however, complete tumor eradication was not observed.The tumor volume observed after 24 days was approximately 400 mm 3 .Significant therapeutic effect of R848 is observed only when R848 is administered using a smart drug delivery platform.Our finding suggested that a dual-targeted nanosystem combined with R848, MIP-3α, and DOX containing an equivalent dose of R848 (1 mg/kg) injected four times in 4T1 tumor-bearing mice suppress tumor volume below 100 mm 3 .
These results further supported that the nanosystem, following intratumoral administration, improved R848 bioavailability in the tumor microenvironment.
Clinical application of DC-based vaccine is limited despite activation of tumor-specific T cells. 37Numerous clinical trials have demonstrated the success of DC-based vaccines in phase I and phase II clinical trial; however, in phase III clinical trial, poor efficacy was observed. 38One of the major reasons underlying the poor success rate is poor migration of DCs in the lymph node. 39Here, we introduced chemokine therapy to solve the issue regarding poor infiltration of immune cells. 40The chemotaxis assay performed in our study demonstrated that MIP-3α incorporated in our nanosystem caused migration of RAW264.7 macrophages, which demonstrated that poor infiltration of immune cells or antigen-presenting cells could be solved by introducing chemokines.We also observed the maximum percentage of infiltrated cytokines in the anti-PD-L1-DOX-R848-MIP-3α/ TKNP-treated tumor tissue.Thus, we believed that the nanosystem constructed by incorporating MIP-3α caused the recruitment of immature DCs in the tumor microenvironment, further increasing cellular immunity against breast cancer. 41cal injection provides better T cell priming in the tumor microenvironment compared to systemic injection. 42In cancer immunotherapy, local injections utilize tumor cells as its own vaccine and help to exert a systemic immune response against tumor cells. 43Additionally, local injection of immune-stimulating moieties enhances tumor antigen recognition and also ensures access to the cytotoxic T cells infiltrated in the tumor tissue. 44In a study by Song et al., 45  Characterization of thioketal polymer was performed using NMR (JEOL NMR, ECZ 500 R; JEOL Ltd., Tokyo, Japan).

| Construction and characterization of DPEMA
PEMA was obtained by the hydrolysis of PEMAnh (poly[ethylene-altmaleic anhydride]; Sigma-Aldrich Corp.).The insoluble PEMAnh was converted to soluble PEMA by mixing PEMAnh (17 mg/ml) in deionized water, and the solution was heated at 60 C for 6 h.Lyophilization was performed to obtain PEMA powder.A nucleophilic addition reaction for 18 h was performed to obtain DPEMA following the reaction between PEMA and dopamine.The organic solvent was removed via dialysis and obtained DPEMA was analyzed using NMR.
Characterization of DOX/R848/MIP-3α-co-loaded TKNP was done by DLS to measure hydrodynamic diameters, zeta potentials, and PDI.Similarly, TEM was used to trace the morphology and particle size.The DOX amount was assessed based on the calibration curve obtained from a fluorescence spectrometer with emission at 580 nm and excitation at 480 nm.Similarly, R848 concentration was measured based on the calibration curve obtained from UV spectroscopy by measuring the absorbance at 254 nm.Furthermore, the MIP-3α content loaded in TKNP was evaluated using Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA).
Similarly, LC and EE were calculated as follows: MIPÀ3α loaded in TKNP The actual amount of TKNP To analyze the ROS-responsive degradation properties of TKNP, TEM was used to monitor the change in the morphology and size following incubation under oxidative stress conditions.Briefly, TKNP was incubated in PBS (pH 7.4) supplemented with H 2 O 2 (0.1 mM) for 0, 6, and 12 h.Next, the size and morphological features were observed under TEM.

| Tailoring of anti-PD-L1 antibody on TKNP using DPEMA
DPEMA used as a surfactant during TKNP synthesis provides an active site for the conjugation of anti-PD-L1 antibody. 15To conjugate anti-PD-L1 antibody (Bio X Cell, Lebanon, NH), TKNP (1 mg) was incubated with different concentrations of anti-PD-L1 antibody (30, 60, 90, 120, and 150 μg/ml) suspended in NaHCO 3 buffer (pH 8.5).Following incubation for 1 h, repeated purification was conducted at 15,000 Â g for 10 min at 4 C. To determine the conjugated amount of antibody on TKNP surface, Pierce BCA protein assay kit (Thermo Fisher Scientific) was used.CE were calculated using the formulae: CE % ð Þ¼ Amount of anti À PD À L1 antibody conjugated to TKNP Initial amount of anti À PD À L1 antibody used Â 100 Similarly, DLS was used to monitor the mean hydrodynamic diameter, PDI, and zeta potential of the anti-PD-L1-TKNP.Additionally, serum cytokine level was measured on day 15 and 20.
Cytokines such as TNF-α and IL-6 were evaluated using a TNF-α ELISA kit (BioLegend, San Diego, CA) and an IL-6 Elisa kit (BD Biosciences) as per the manufacturer's instructions.

| Statistical analysis
Data were expressed as mean ± SD, and statistical significance was measured using Student's two-tailed t-test.Statistical significance was set at p < 0.05.

| CONCLUSIONS
We reported a novel synthesis of PD-L1-targeted ROS-responsive

1 2 |
Schematic illustration depicting the anti-PD-L1-DOX-R848-MIP-3α/TKNP-mediated activation of cellular immunity against breast cancer.A mouse xenograft model was developed to evaluate the therapeutic efficacy of anti-PD-L1-DOX-R848-MIP-3α/TKNP following intratumoral administration.In the tumor microenvironment, degradation of TKNP occurs due to the high ROS level.Release of DOX from TKNP causes ICD, chemokines enhance the migration of dendritic cells, and R848 causes maturation of dendritic cells.Thus, the synergy between the ICD phenomenon and the migration and maturation of dendritic cells results in the activation of T cells.Meanwhile, activated T cells release cytokine causing tumor eradication CDCl 3 ) of the thioketal polymer suggesting the 6H (methyl group) and 4H group further depicted the proton adjacent to the thioketal group.The proton peak at approximately 7-8 ppm suggested an aromatic ring.Figure 2c shows the 13 C-NMR results of thioketal polymer with δ ppm @ 32.01, 35.23, 36.12, and 43.26. 1 H-NMR (Figure 2d) further demonstrates the successful construction of dopamine-conjugated poly(ethylene-alt-maleic acid) (DPEMA).The 1 H-NMR (D 2 O) of DPEMA showed a peak at δ (ppm) 6.61 ( 1 H), 6.69 ( 1 H), and 6.76 ( 1 H), suggesting the presence of aromatic proton and carbon, confirming the successful synthesis of DPEMA.F I G U R E 2 (a) Synthetic scheme for the synthesis of ROS-responsive immunomodulatory nanosystem.(b) 1 H NMR and (c) 13 C NMR of thioketal polymer.(d) 1 H NMR of D-PEMA.(e) Hydrodynamic diameter, TEM image, and zeta potential (f) measurement of TKNP.(g) Morphological characterization showing the degradation of TKNP after incubation with 0.1 mM H 2 O 2 .(h) Amount of anti-PD-L1 antibody conjugated on TKNP.(i) CE of different amounts of anti-PD-L1 antibody conjugated on TKNP.The release profile of DOX (j) and R848 (k) from anti-PD-L1-DOX/R848 at an acidic pH (5.0), acidic pH (5.0) + 0.1 mM H 2 O 2, and neutral pH (7.4).Data are expressed as the mean ± SD 2.Construction and characterization of DOX, MIP-3α, and R848-laden TKNP Confocal laser scanning microscopy (CLSM) demonstrated that compared to TKNP alone, anti-PD-L1-TKNP showed increased cellular internalization of TKNP (strong green fluorescence) in the MDA-MB-231 cell line.Furthermore, in the BT-20 cell line, there was no significant difference in cellular internalization between TKNP and anti-PD-L1-TKNP (Figure 3b).Internalization of nanoparticles and DOX was further evaluated by generating a 3D model of MDA-MB-231 and BT-20 cell lines (Figure 3c).CLSM demonstrated that compared to the control, DOX/TKNP showed significant internalization of DOX (red fluorescence) and nanoparticles (green fluorescence) both in MDA-MB-231 and BT-20 cell lines.However, following anti-PD-L1-DOX/TKNP treatment, MDA-MB-231 showed significant internalization of nanoparticles and DOX compared to DOX/TKNP alone.However, there was no significant difference in the cellular internalization of DOX and nanoparticle between targeted and nontargeted nanoparticles in MDA-MB-231 and BT-20 cell lines.Furthermore, the competitive receptor binding assay showed decrease in fluorescence intensity of DOX and coumarin-6 following 4 h incubation with anti-PD-L1 antibody pretreatment + anti-PD-L1-DOX-TKNP group compared to anti-PD-L1-DOX-TKNP (Figure Incubation with DOX/TKNP further increased CRT expression in both MDA-MB-231 (6.1-fold; p < 0.001) and BT-20 (4.9-fold; p < 0.001) cell lines compared to free DOX.CRT expression significantly increased following anti-PD-L1-DOX/TKNP treatment (1.8-fold; p < 0.01) in the MDA-MB-231 cell line compared to the DOX-TKNPtreated group.However, there was no significant difference in CRT expression in the BT-20 cell line between nontargeted and targeted groups.
Nanoparticles were labeled with an equivalent concentration of Cy5.5.The tumor retention assay demonstrated that anti-PD-L1-TKNP displayed longer retention in the tumor zone up to 12 h (1.4-fold; p < 0.05) and 24 h (1.3-fold; p < 0.05) compared to the TKNP alone (Figure 5a,b).Maximum retention of targeted nanoparticles in the tumor zone compared to nanocarrier alone is attributed to the PD-L1 receptor-mediated endocytosis compared to the enhanced permeability and retention effect alone.

2. 12 |
Investigation of ICD phenomenon, DOX retention, and DC maturation study After confirming the better anti-cancer effect of anti-PD-L1-DOX-R848-MIP-3α/TKNP in the in vitro condition, we investigated the ICD phenomenon in dying tumor cells.Generally, in ICD, dead cancer cells are eventually converted into a vaccine for activating cytotoxic T cells against tumor cells. 4Thus, we investigated the ICD phenomenon after sacrificing mice on day 15.Tumors were excised from the mice on day 15, and the tumor retention of DOX and DOX-induced ICD phenomenon was examined by immunofluorescence staining of the tumor section.To determine the ICD on day 15, we measured CRT expression in the tumor tissue (Figure 5k).Compared to other groups, anti-PD-L1-DOX-R848-MIP-3α/TKNP increases CRT exposure (green fluorescence) and provides maximum retention of DOX (red fluorescence) in the tumor tissue.Thus, maximum retention of DOX in the tumor tissue offered by anti-PD-L1-DOX-R848-MIP-3α/TKNP resulted in robust ICD induction efficacy.Furthermore, we also measured CRT and HMGB1 expression in the tumor section of the mice sacrificed on day 20 to further confirm the ICD (Figure 5l).Anti-PD-L1-DOX-R848-MIP-3α/TKNP caused maximum exposure of CRT and HMGB1 release in the tumor zone which is further confirmed by the strong green fluorescence.

1 |
after the systemic injection of R848-loaded bismuth selenide nanocage following NIR irradiation to 4T1 tumor-bearing mice, the amount of CD8 + T cells infiltrated in the tumor tissue was below 5%.Thus, in our study, we aimed to increase the percentage of CD8 + T cells in the tumor tissue by locally injecting anti-PD-L1-DOX-R848-MIP-3α/ TKNP in 4T1 tumor-bearing mice.We demonstrated maximum priming of T cells in the tumor tissue with CD8 + T cells (9.5 ± 3.2%) and CD4 + T cells (18.4 ± 7.6%) suggesting local injection of R848 embedded in a smart nanosystem increases cytotoxic activities of T cells.Due to maximal tumor deposition, significant active and passive targeting, and ROS-responsive release properties, TKNP combined with immunotherapeutic moiety caused maximal tumor eradication with low side effects.The anti-PD-L1-DOX-R848-MIP-3α/TKNP has significant potential to enhance the prognosis of patients with refractory tumor in a clinical setting and has a wide range of clinical applications.Additionally, targeted delivery of immunoadjuvants to endogenous immune cells and priming of immune cells in the tumor microenvironment highlighted the potential of this strategy for additional investigation in overcoming immunological tolerance.This study also further highlighted the scope of immune checkpoint blockade therapy which could be due to the dissociation of an anti-PD-L1 antibody from our nanocarrier.In addition, the therapeutic payload encapsulated in our nanosystem can function for synergistic effect as high infiltration of DCs in the tumor microenvironment due to MIP-3α can boost the therapeutic effect of R848 on triggering DC maturation.Furthermore, we can also broaden the scope of this study by studying the interaction of these active drugs with the several cell type present in the tumor microenvironment.4| MATERIALS AND METHODS4.Synthesis and characterization of ROSresponsive thioketal polymerThioketal polymer was constructed using an acetal exchange reaction following stepwise polymerization.Briefly, 4,4 0 -bis(mercaptomethyl) biphenyl (16 mg/mL; Sigma-Aldrich Corp., St. Louis, MO) dissolved in toluene (Sigma-Aldrich Corp.) was further mixed with 2,2-DMP (250 μl; Sigma-Aldrich Corp.).Next, the mixture was stirred at 75 C and p-toluenesulfonic acid (2 mg; Sigma-Aldrich Corp.) dissolved in ethyl acetate (250 μl) to initiate the reaction.After 1 h of reaction, a mixture of DMP (500 μl) in toluene (10 ml) was kept at an interval of every 30 min for 10 h for polymerization.Precipitation with cold hexane (Sigma-Aldrich Corp.) was performed to obtain thioketal polymer.